18 Generation of ATP

Dr. Ramesh Kothari

Biochemical mechanisms of generating ATP

In metabolisms, ATP is generated by two fundamental different biochemical mechanisms:

1. Substrate level phosphorylation, and
2. Electron transport chain.

Substrate level phosphorylation

• In substrate level phosphorylation, ATP is formed from ADP by transfer of a high energy phosphate group from an intermediate of a fueling pathway.
• The following reaction serve as an example

• As a consequence of the removal of a molecule of water, the low- energy ester linkage of phosphate in 2- phosphoglyceric acid is converted to the high energy enol linkage in phosphoenol pyruvic acid.

• This high- energy linked phosphate can then then be transferred to ADP, the consequence of which is generation of a molecule of ATP.

Generation of ATP by Electron Transport

• In a number of different mode of microbial metabolisms including respiration and photosynthesis, ATP is generated by transporting electron through the chain of carrier molecules with fixed orientation in a cell membrane.

• Although the complexity and component of electron transport chain vary, they have a certain common features:

• The component of chain are carrier molecules capable to undergoing revercible oxidation and reduction ; each member of chain is capable being reduced by reacting with the carrier molecules that precedes it and oxidized by the carrier that follows it.

• In any specific example of an electron transport chain, certain members transport hydrogen atoms while other transport only electron.

• The orientation of carrier in the cell membrane is such that hydrogen carriers transport in the direction toward the outside of the cell and electron carrier transport toward the inside.

• Thus, at each conjunction in the chain of a hydrogen carrier and an electron carrier, a proton is transported out of the cell.

• The cell membrane is otherwise impermeable to proton ; as consequence electron transport traps a portion of the chemical energy released by the net reaction of the chain in the form of gradient across the membrane of proton and electric charge.

• Such a gradient termed as proton motive force (Δp) is form of potential energy capable of doing work : it drives certain permease system that concentrate externally supplied substrate within the cell; it provides the energy for flagellar-mediated cell motility and its drives the energy requiring synthesis of ATP from ADP.

• The synthesis of ATP at the expense of protonmotive force is catalyzed by complex membrane bound enzyme , ATP phosphohydrolase ( some time termed as ATP ase ) composed in all bacterial studied , of two multicomponant protein BF0 and BF1.

• The subunit composition and membrane insertion of BF0 and BF1 are shown in figure.

• The α and β subunit of BF1are arranged alternately to form a hollow hexagon, the central hole of which contain the γ subunit associated with other subunit δ and ε.

• Thus BF1 probably has the subunit structure α3β3γδε .

• The α and β subunit form the catalytically active portion of structure ,the site where ATP is synthesized from ADP and inorganic phosphate ;the γ,δ and ε subunit form a proton translocating stalk and gate that bring to the active site at the proper rate the proton that drive reaction.

• The peptide form a proton channel through the membrane they are hihly hydrophobic accounting for their intra membrane location

• ATP phosphohydrolase catalyzed a reversible reaction .ATP can be synthesize at a expense of proton motive force ,or in certain case a proton motive force can be established at the expense of intracellular ATP.

Schematic representation of the subunit composition and membrane insertation of

Value of E0’ for components in electron transport chains

• In order for an electron transport chain to function ,there must be a gradient of susceptibility to oxidation; i.e each component must be capable of being reduced by reduced form of previous component and oxidized by the oxidized form of the subsequent component in the chain.

• The relative susceptibility of a substance to oxidation or reduction can be described quantitatively in terms of its electrode potential or reduction potential ;this is the relative voltage required to remove an electron from H2. Thus standard reduction potential is that of hydrogen electrode

• Which is assigned an arbitrary value of 0.0 volts under standard condition . At the pH 7.0,near which most biological reaction occurs, the potential of hydrogen electrode is -0.42 V.

• The symbol E0’ designates electrode potentials measured under these condition .

• Knowing E0’ value of two half reaction , the free energy changed of a coupled reaction can be calculated from the relationship

• Where ΔG’0 is the free energy change at pH 7.0 ;n is the number of electron transferred ;F is the faraday and ΔE’0 is the algebraic difference between the potential of the two half reaction.

• For example the reduction of oxygen by hydrogen as (H2 +1/2 O2àH2O) can be divided in to two half reactions.

• The energy change can be calculated to be

• For a typical biology oxidation , for example , the oxygen- linked oxidation of NADH (ΔE’0 = -0.32V), the analogous calculation shows a free energy change of -52,400 cal, not significantly different from that for oxidation of hydrogen.

• The carrier in an electron transport chain participate in a series of reaction of increasing ΔE’0 values , between that of the primary electron donor and terminal electron acceptor.

• The position on the E’0 scale of several typical electron carrier ,primary electron donor, and terminal electron acceptor are shown in figure.

The Componants of Electron Transport Chain

• The electron transport chain of aerobic chemoheterophs are the most thoroughly studied.

• Those involved in the oxidation of organic compound always contain four different classes of molecules.

• Two classes, the flavoprotein and quinines ,are hydrogen carrier; the others,the iron sulphur protein and cytochromes are electrone carriers.

• Flavoprotein have a yellow-colored prosthetic group derived biosynthetically from riboflavin (vitamin B12).

• The prosthetic group may be either flavin mononucleotide (FAD); both possess the same active site capable of undergoing reversible oxidation and reduction by donating or accepting two hydrogen atom respectively.

• The flavo proteins are members of a large class and differ widely with respect to their E’0 values.

• Some are active in the primary dehydrogenation of organic substrate (e.g succinate) ;other participate as hydrogen carriers within an electron transport chain; and still other react directly with molecular oxygen with the formation of H2O2.

• Most electron transport chain contain either ubiquinone , a substituted benzoquinone, or menaquinone , a substituted nepthaquinone.

• The former occur most frequently in Garm negative bacteria and in the mitochondria of eukaryotes, and the latter most frequently in Gram positive bacteria.

• A few facultative anaerobes, including, E.Coli ,contain both quinines but tend to use ubiquinone for aerobic respiration .

• On reduction they accept two hydrogen atom to form the corresponding quinol.

• The iron sulfur protein contain two, four or eight atoms of labile sulfur, so called because they are released as H2S by strong acids.

• The iron atoms in 2 Fe-S protein are held in a lattice composed of four atoms of cysteine sulfur and the two labile sulfur atoms.

• The iron atoms in the 4Fe-4S proteins interact with four labile sulfur atoms to form a cube that is held in place within the protein by four cysteine residue.

• The 8Fe-8S protein contain two active centers identical to those found in 4Fe-4S protein, thus enabling them to accept on reduction, two electrons while 2Fe-2S and 4Fe-4S protein can accept only one.

• The cytochromes belong to the class of heme protein, a class that also includes hemoglobin and catlase.

• All have one or more prosthetic groups derived from heme, a cyclic tetrapyrrole with an atom of iron chelated within the ring system.

• Electron transfer by the cytochromes involves a reversible oxidation of this iron atom

• In an analogous way to the oxidation of an iron atom in the iron –sulfur proteins.

• The cytochromes have characteristic absorption band in the redused state that permit recognition of the several different members of the class, which are designated by terminal latter (e.g.,cytochrome c).

• There are four types of heme prosthetic groups known (a,b,c,and d) that differ principally in the nature of the substituent group at the periphery of the heme.

• Individual cytochromes contain one or more of these groups in various combinations.

• The molecular weights and reduction potentials of cytochromes vary widely.

• Cytochrome contentof bacteria is quite variable among species and environments.for example, an aerobically grown bacterium will have a different cytochrome composition from that of the same strain grown anaerobically or even grown with a restricted supply of oxygen.

• The presence of a c- type cytochrome in a particular bacterium is correlated with the outcome of anempirical procedure , the oxidase test,whichhas considarble , diagnostic importance in identification of aerobic bacteria.

• The test is performed by putting a small quantity of bacteria ona piece of filter paper soaked in a dye ; either dichlrophenol indophenols or N,N dimethyl-p-phenylene – diamine.

• These dyes which are colorless in the reduced form by oxidase –positive species(which lake a c-type cytochrome).

Arrangement of Electron Transport Chain in Membrane

• As stated bacteria are quite variable with respect to their cytochrome content ; they are also variable with respect to other constituents of electron transport chain.

• A proposal for the aerobic respiratory chain in Esherichia coli is shown in figure.

• NADH in the cytoplasm generated by a reaction in a fueling pathway transfers on the inner aspect of the membrane two hydrogens atoms toa flavoprotein embeded within the membrane.

• On the outer aspect of the membrane the flavoprotein transfers two electrons to an iron sulfurprotein thereby releases them out side when it transfer twoelectron toa b-type cytochrome.

• Finally, the cytochrome b transfer its electrons to another cytochrome that is termed a cytochrome oxidase because it has the capacity to transfer electrons to molecular oxygen in a reaction on the inner surface that has H2O as a product.

• It will be noted that for each pair of electron transported through the chain four protons are pumped out of the cell, thus creating a proton motive force that can be used to do cellular work , for example ATP synthesis ,through the membrane bound ATP phosphohydrolase .

• It will also be noted that composition of the chain varies with the avilibility of oxygen.

• When fully aerobic ,cytochrome b550 serve as terminal electron carrier and cytochrome o as the terminal oxidase.

• When the supply of oxygen is limited cytochrome b558 and d play the corresponding roles.

References

Books

• Roger Y. Stanier, John L. Ingraham, Mark L. Wheelis, Page R. Painter. Genral Microbiology, Fifth edition 1995.
• Clayton, R.K.,Photosynthesis: Physical mechanisms and chemical petterns.Cambridge: Cambridge university press, 1980.
• Gottschalk,G.,Bacterial Metabolisms. New York, Heidelberg, and Berlin:springer-Verlag,1979.
• Ingraham,JL.,O. Malloe and F.C Neidhardt, Growth of the bacterial cell.Sunderland,Mass.: Sinauer associates, INC.,1983.
• Jones,C,W.,Bacterial respiration and photosynthesis Washington,D.C.:American society for microbiology,1982.
• Lehninger,A.L., Principles of Biochemistry.New York:Worth publishers,1982.
• Mandestam,J., and K.Mcquillen, Biochemistry of Bacterial growth. New York: John wiley,1982.

Web

• http://www.angelfire.com/ex/firstperiodbiology/